CN107916105A - It is a kind of to be used to detect red fluorescence carbon quantum dot of internal pH and preparation method thereof - Google Patents

Abstract

The present invention provides a kind of for detecting red fluorescence carbon quantum dot of internal pH and its preparation method and application.Preparation method：(1) p-phenylenediamine for weighing 0.1 0.2mmol is dissolved in 0.075 0.15mol ethylenediamines, obtains weak yellow liquid；(2) above-mentioned solution is placed in micro-wave oven, 5~25min is reacted under 20%~100% power, reaction stops postcooling to room temperature, adds 5mol/L HCl to there is gas effusion；(3) above-mentioned resulting solution is put into the bag filter of 500 1000Da, then bag filter is put into the container equipped with water, dialysed two days, that is, obtain pure carbon quantum dot aqueous solution, the cotton-shaped carbon quantum dot of pale pink is obtained after being freeze-dried.Carbon quantum dot obtained by the present invention has the features such as long transmitting, high quantum production rate, good biocompatibility；Carbon quantum dot has pH very sensitive response, and can penetrate nuclear membrane and enter nucleus, hence into whole cell, reaches the effect for detecting whole cellular pH.

Description

A red fluorescent carbon quantum dot for detecting intracellular pH and its preparation method

technical field

The invention relates to nanometer materials, in particular to a red fluorescent carbon quantum dot for detecting the pH in the whole cell, a preparation method and application thereof.

Background technique

As an important metabolic and intracellular parameter, cellular pH plays a key role in regulating the physiological and pathological processes of many cells. Abnormal pH will cause dysfunction of cells and tissues in the body, inhibit the activity of enzymes and proteins, reduce the body's immunity, and eventually lead to diseases such as inflammation, cancer, and Alzheimer's disease. In addition, changes in cellular pH are closely related to apoptosis. In the initial stage of apoptosis, cancer cells generally cause intracellular acidification, and intracellular acidification has become an important early feature of apoptosis. Therefore, sensitive and accurate real-time in situ monitoring of cellular pH is helpful for understanding cellular physiological and pathological processes at the molecular level.

Fluorescence spectroscopy has the advantages of high sensitivity, good selectivity, fast response, high signal-to-noise ratio, and easy operation. Combined with fluorescence microscopy imaging technology, pH fluorescent probes can monitor the dynamic distribution and regional changes of intracellular pH in real time and in situ. Showing its unique nature of high temporal and spatial resolution.

As a new type of carbon nanomaterial, carbon quantum dots have excellent properties such as good water solubility, biocompatibility, low toxicity, photostability, easy functionalization and photobleaching resistance in bioimaging, biosensing, and pharmaceuticals. Transportation and many other fields have good application prospects. In recent years, synthesized carbon quantum dots usually emit blue and green fluorescence, which limits their applications in the biological field. In addition, in terms of cell imaging, most of the current fluorescent nanoprobes are limited to the cell matrix in terms of cell imaging, and cannot penetrate the nuclear membrane into the nucleus. Therefore, it is of great significance to realize carbon quantum dots that emit red light efficiently and can enter the nucleus through the nuclear membrane. In view of the above problems, the present invention provides a method for synthesizing carbon quantum dots by microwave method using p-phenylenediamine and ethylenediamine as raw materials, which will be finally used for the detection of pH in biological cells.

Contents of the invention

The purpose of the present invention is to provide a red carbon quantum dot capable of penetrating the nuclear membrane and entering the nucleus to detect the pH of the whole cell, its preparation method and application.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

A preparation method of red fluorescent carbon quantum dots for detecting intracellular pH

(1) Weigh 0.1-0.2 mmol of p-phenylenediamine and dissolve it in 0.075-0.15 mol of ethylenediamine to obtain a light yellow liquid;

(2) Put the above solution in a microwave oven, react at 20% to 100% power for 5 to 25 minutes, cool to room temperature after the reaction stops, add 5mol/L HCl until gas escapes;

(3) Put the above obtained solution into a 500-1000 Da dialysis bag, then put the dialysis bag into a container equipped with water, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain a light Pink flocculent carbon quantum dots.

The obtained carbon quantum dots were dissolved in PBS buffer solution and added to the culture medium of A549 cells, incubated for 6 hours, and then the imaging state of A549 cells was observed by laser confocal microscope. When the carbon quantum dots are at a pH of 5.8, the cells show bright red fluorescence; when the carbon quantum dots are at a pH of 7.4, the cells show very weak fluorescence. It shows that the carbon quantum dot can be used as a detection reagent for the detection of the pH of the whole cell including the nucleus.

Compared with the prior art, the present invention has the following advantages:

The carbon quantum dots prepared by the present invention have the characteristics of long emission, high quantum yield, good biocompatibility and the like;

The carbon quantum dot prepared by the invention has a very sensitive response to pH, and can penetrate the nuclear membrane and enter the nucleus, thereby entering the whole cell, so as to achieve the effect of detecting the pH of the whole cell.

Description of drawings

Fig. 1 is the transmission electron micrograph of the fluorescent carbon quantum dot prepared by the embodiment of the present invention 1;

Fig. 2 is the ultraviolet absorption spectrum and the fluorescence excitation-emission spectrum of the fluorescent carbon quantum dot prepared in Example 1 of the present invention;

Fig. 3 is the fluorescent carbon quantum dot prepared in Example 1 of the present invention at 592nm place fluorescence intensity changes curve (1.4-7.4) with pH value;

Fig. 4 is a laser confocal display diagram of fluorescent carbon quantum dots prepared in Example 1 of the present invention entering the whole cell under the conditions of pH = 5.8 and pH = 7.4 respectively.

Detailed ways

Example 1

A preparation of red fluorescent carbon quantum dots for detecting intracellular pH in this embodiment comprises the following steps:

(1) Weigh 0.2 mmol of p-phenylenediamine and dissolve it in 0.15 mol of ethylenediamine to obtain a light yellow liquid;

(2) Place the above solution in a microwave oven, react at 20% power for 20 minutes, cool to room temperature after the reaction stops, add 5mol/L HCl until gas escapes;

(3) Put the above obtained solution into a dialysis bag of 500-1000Da, then put the dialysis bag into a 1000ml beaker, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain light pink flocs carbon quantum dots. The quantum yield of the carbon quantum dots was measured to be 14%.

Dissolve the obtained carbon quantum dots in PBS buffer solutions with pH=5.8 and pH=7.4 respectively, make 50μg/ml solutions, add them to the culture medium of A549 cells, incubate for 6h, and then observe through laser confocal microscope Imaging status of A549 cells.

Structural characterization of the carbon quantum dots synthesized above was performed, as shown in Figure 1. The carbon quantum dots are uniform spherical in shape, and the particle size is about 4.6nm.

The spectral properties of the carbon quantum dots synthesized above were studied, as shown in Figure 2. The optimum excitation wavelength and the optimum emission wavelength of the carbon quantum dot are 535nm and 592nm respectively.

The above-mentioned synthesized carbon quantum dots exhibit fluorescence changes at different pHs, as shown in Figure 3. The carbon quantum dot presents an S-shaped curve at pH=2.0-9.0, the pKa is 5.8, and the linear range is 4.5-7.0.

The conditions of the above-mentioned synthesized carbon quantum dots entering the cells were observed through laser confocal experiments, as shown in Figure 4. When the carbon quantum dots are at a pH of 5.8, the cells exhibit bright red fluorescence, and it can be clearly observed from the imaging image of the cells that the entire cell nucleus exhibits bright red fluorescence, which shows that the above-mentioned synthesized carbon quantum dots have successfully into the nucleus. When the carbon quantum dots were at a pH of 7.4, the cells showed very weak fluorescence. It shows that the carbon quantum dots can be successfully used to detect the pH of the whole cell including the nucleus.

Example 2

(1) Weigh 0.1 mmol of p-phenylenediamine and dissolve it in 0.15 mol of ethylenediamine to obtain a light yellow liquid;

(2) Place the above solution in a microwave oven, react at 40% power for 20 minutes, cool to room temperature after the reaction stops, add 5mol/L HCl until gas escapes;

(3) Put the above obtained solution into a dialysis bag of 500-1000Da, then put the dialysis bag into a 1000ml beaker, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain light pink flocs carbon quantum dots. The quantum yield of the carbon quantum dots was measured to be 12%.

Example 3

(1) Weigh 0.2 mmol of p-phenylenediamine and dissolve it in 0.075 mol of ethylenediamine to obtain a light yellow liquid;

(2) Place the above solution in a microwave oven, react at 60% power for 15 minutes, cool to room temperature after the reaction stops, add 5mol/L HCl until gas escapes;

(3) Put the above obtained solution into a dialysis bag of 500-1000Da, then put the dialysis bag into a 1000ml beaker, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain light pink flocs carbon quantum dots. The quantum yield of the carbon quantum dots was measured to be 10%.

Example 4

(1) Weigh 0.2 mmol of p-phenylenediamine and dissolve it in 0.15 mol of ethylenediamine to obtain a light yellow liquid;

(2) Place the above solution in a microwave oven, react at 80% power for 10 minutes, cool to room temperature after the reaction stops, add 5mol/L HCl until gas escapes;

(3) Put the above obtained solution into a dialysis bag of 500-1000Da, then put the dialysis bag into a 1000ml beaker, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain light pink flocs carbon quantum dots. The quantum yield of the carbon quantum dots was measured to be 11%.

Example 5

(1) The p-phenylenediamine that takes 0.2mmol is dissolved in 0.15mol l ethylenediamine, obtains pale yellow liquid;

(2) Put the above solution in a microwave oven, react at 100% power for 5 minutes, cool to room temperature after the reaction stops, add 5mol/L HCl until gas escapes;

(3) Put the above obtained solution into a dialysis bag of 500-1000Da, then put the dialysis bag into a 1000ml beaker, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain light pink flocs carbon quantum dots. The quantum yield of the carbon quantum dots was measured to be 8%.

comparative example

(1) Weigh 0.2 mmol of p-phenylenediamine and dissolve it in 0.15 mol of ethylenediamine to obtain a light yellow liquid;

(2) Put the above solution in a microwave oven, react at 20% power for 5 minutes, and cool to room temperature after the reaction stops;

(3) Put the above obtained solution into a dialysis bag of 500-1000Da, then put the dialysis bag into a 1000ml beaker, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain light pink flocs carbon quantum dots with a quantum yield of 3%.

Compared with the carbon quantum dots obtained in Example 1, the obtained carbon quantum dots had significantly lower quantum yield and yield, and their properties were unstable.

Claims (5)

1. a preparation method for detecting the red fluorescent carbon quantum dot of intracellular pH, is characterized in that, comprises the steps: (1) Weigh 0.1-0.2 mmol of p-phenylenediamine and dissolve it in 0.075-0.15 mol of ethylenediamine to obtain a light yellow liquid; (2) Put the above solution in a microwave oven, react at 20% to 100% power for 5 to 25 minutes, cool to room temperature after the reaction stops, add 5mol/L HCl until gas escapes; (3) Put the above obtained solution into a 500-1000 Da dialysis bag, then put the dialysis bag into a container equipped with water, and dialyze for two days to obtain a pure aqueous solution of carbon quantum dots, which is freeze-dried to obtain a light Pink flocculent carbon quantum dots. 2. the preparation method of red fluorescent carbon quantum dot as claimed in claim 1 is characterized in that, in step (1), the amount of p-phenylenediamine is 0.2mmol, and the amount of ethylenediamine is 0.15mol. 3. the preparation method of red fluorescent carbon quantum dot as claimed in claim 1 is characterized in that, the microwave oven power in the step (2) is 20%, and the reaction time is 20min. 4. The red fluorescent carbon quantum dots for detecting intracellular pH prepared by the method according to claim 1, 2 or 3. 5. the application of red fluorescent carbon quantum dot as claimed in claim 4 in the reagent of preparation detection intracellular pH.